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Figure 1. Saturation binding for CCR7. 10 µg/well CCR7 Membrane Preparation was incubated with increasing amount of [¹²⁵I]- MIP-3β in the ...

Figure 2. Competition binding for CCR7. CCR7 Membrane Preparation (10 µg/well) was incubated with 0.1 nM [¹²⁵I]-MIP-3β and increasing co ...

ChemiSCREEN™ CCR7 Membrane Preparation, recombinant

Description:

Trade Name:

ChemiScreen

Product Overview:

Full-length human CCR7 cDNA

Background Information:

CCR7 is a GPCR that binds to CC chemokine ligands MIP-3beta (ELC/CCL19) and 6Ckine (CCL21)1. These ligands are expressed in the secondary lymphoid organs, and binding to CCR7 expressed in naïve T cells, B cells and dendritic cells directs migration of these cells to sites of antigen presentation2. Inhibition of CCR7/ligand interactions inhibits contact sensitivity, delayed type hypersensitivity, and graft vs host disease in experimental models2,3. In addition, CCR7 expression by breast cancer, melanoma and other malignant cells are associated with lymph node metastasis4,5. Chemicon's CCR7 Membrane Preparations are ideal for screening for inhibitors of CCR7/ligand interactions. The CCR7 membrane preparations bind to MIP-3beta with a Kd of approximately 0.2 nM. At a [125I]-MIP-3beta concentration of 0.1 nM, a signal to background ratio of greater than 8-fold is typically obtained.

Application Notes:

Radioligand binding assay and GTPγS binding

Species Reactivity:

Human

Usage Statement:

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

Host Cells:

Chem-2, a suspension mammalian cell line without any endogenous CCR7 expression.

Cell Type:

Chem-2

Gene Symbol:

CCR7
CMKBR7
EBI1
CD197
CDw197
CCR-7
BLR2
EVI1
CC-CKR-7

Format:

Membranes

Presentation:

Membranes were adjusted to 0.5 mg protein/ml in 50 mM Tris pH 7.4, 10% glycerol and 1% BSA, rapidly frozen, and stored at -80°C, sufficient for 200 microassays per vial.

Quality Assurance:

SPECIFICATIONS: 1 unit = 10 µg membrane preparation
Bmax: 3.3 pmol/mg
Kd: 0.2 nM

	10 µg/well
Signal:Background	10.2
Specific Binding (cpm)	8362

Table 1. Signal:background and specific binding values obtained in a competition binding assay with CCR7 membrane preparation.

Protein or Enzyme Type:

Membrane Preparations

Incubation Conditions:

Membranes are mixed with radioactive ligand and unlabeled competitor (see Figures 1 and 2 for concentrations tested) in binding buffer in a nonbinding 96-well plate, and incubated for 1-2 h. Prior to filtration, a GF/C 96-well filter plate is coated with 0.33% polyethyleneimine for 30 min, then washed with 50mM HEPES, pH 7.4, 0.5% BSA. Binding reaction is transferred to the filter plate, and washed 3 times (1 mL per well per wash) with Wash Buffer. The plate is dried and counted.
Binding buffer: 50 mM Hepes, pH 7.4, 5 mM MgCl₂, 1 mM CaCl₂, 0.2% BSA, filtered and stored at 4°C
Radioligand: [¹²⁵I] MIP-3β (Perkin Elmer #NEX370 )
Wash Buffer: 50 mM Hepes, pH 7.4, 500mM NaCl , 0.1% BSA, filtered and stored at 4°C.
One package contains enough membranes for at least 200 assays (units), where an unit is the amount of membrane that will yield greater than 7-fold signal:background with [¹²⁵I]-labeled MIP-3β at 0.1 nM

Species:

Human

Storage Conditions:

Maintain frozen at -70°C for up to 2 years. Do not freeze and thaw.